CG endocytosis is blocked at an early stage in Flower-deficient CTLs. (A) CLEM composite images of SybKI CTL (top) and Flower KO/SybKI CTL (bottom) in contact with the target cell. CTLs were incubated with target cells for 35 min in the presence of anti-RFP488 (green) in the medium to label endocytic CGs. After fixation, the processed fluorescent SIM image was overlaid with the transmission electron microscopy (TEM) image. Bars, 2 µm. (B) Magnification images from panel A of CTL and target cell contact region. Bars, 2 µm. (C) TEM images with green arrows marked the endocytic events in the SIM images. Bars, 1 µm. (D) Quantitative analysis of endocytic events distribution in SybKI CTLs (control) and Flower KO/SybKI CTLs from the CLEM experiment. The IS is defined as the interface between the CTL and target cells at the contacted membrane. The non-IS region is the remaining part in CTLs including cytoplasm and plasma membrane aside contact site. 147 endocytic events were analyzed from SybKI CTLs (n = 25), and 136 endocytic events were analyzed from Flower KO/SybKI CTLs (n = 26). (E) Quantitative analysis of ultrastructure of endocytic events at the IS corresponding to anti-RFP488 fluorescence green spots. Relative frequency of pit-like structures over total events were shown in the graph from SybKI CTLs (n = 25) and Flower KO/SybKI CTLs (n = 26). Data are given as mean ± SEM; Mann–Whitney U test; **, P < 0.01.